The epitopes for 3 broadly-neutralizing (Nt) MAbs, 2F5, 4E10 and Z13, are located in the membrane proximal region of the HIV-1 envelope protein, gp41 (MPR). Moreover, two of these MAbs neutralize a larger spectrum of HIV-1 primary isolates than any of the other three broadly-Nt MAbs. This makes the MPR a prime site for neutralization, and as such, a prime target for the development of a protective vaccine against AIDS. However, there is also evidence that the MPR-specific Abs produced during natural infection are not neutralizing, though this has not been rigorously tested. It may be that only a certain form of the MPR will elicit Nt Abs. Most or all of the MPR appears to interact with the plasma membrane and NMR studies show that this region forms a helical structure in micelles, but not in aqueous solution Repeated attempts to immunize with engineered peptides or fusion proteins bearing the 2F5 linear epitope have all failed to elicit Nt activity, in face of strong Ab responses against the epitope. Recent structural studies indicate that the epitopes for 2F5 and 4E10 MAbs may comprise both protein and lipid. Thus, we and others have hypothesized that the MPR may elicit Nt Abs if it is presented to the immune system in the context of lipid or the plasma membrane. Moreover, other features, such as other regions of gp41, or specific areas of the cell membrane, may contribute to the structure of the MPR on neutralization-sensitive envelope spikes. Thus, we have designed a panel of 14 different gp41 fragments, and have analyzed their expression. We have begun to show that one of these proteins is expressed on the cell surface, as intended. In this proposal we describe 3 specific aims: (i) to identify gp41 fragments that are located on the cell surface, and to determine whether they form trimers and their relative affinity for MAbs 2F5 and 4E10; (ii) to use the DNA encoding the most promising of these gp41 constructs to immunize rabbits; and (iii) to test the resulting immune sera for their ability to neutralize HIV-1 primary isolates, and to determine if these Nt reactivities are specific for the MPR. Our goal is to determine if Nt Abs can be elicited by the MPR in the context of the cell surface. If successful, we will then use our results to develop a MPR-targeting vaccine. Thus, this RO3 proposal addresses a central public health goal of the NIAID, in determining the validity of the MPR, in the context of the cell surface, as a target for vaccine development. [unreadable] [unreadable]